Parathyroid hormone-related peptide (PTHrP) was discovered as the agent frequently responsible for the humoral hypercalcemia of malignancy. Because PTHrP is made in many tissues, the protein is thought to act often in a paracrine or autocrine fashion, but its normal functions in vivo are poorly understood. Many of the probably multiple postulated paracrine actions of PTHrP may be mediated by the recently cloned receptor that binds both PTHrP and parathyroid hormone (PTH). It is unclear whether and how one receptor can mediate the homeostatic actions of PTH, while also mediating the non-homeostatic, paracrine actions of PTHrP. To study the function of PTHrP in the intact organism, the DNA sequence encoding the PTHrP coding region was deleted from murine embryonic stem (ES cells) and these cells were introduced into blastocysts. The blastocysts developed into chimeric mice. After appropriate matings mice missing both copies of the PTHrP gene were found to have misshapen skeletons and to die at birth. This proposal's first Aim is to characterize the consequences of this PTHrP deficiency. Abnormalities in the proliferation and differentiation of chondrocytes in endochondral bone will be evaluated by determining how PTHrP deficiency alters the genetic program of the growth plate and perturbs the normal kinetics of cellular proliferation and differentiation of chondrocytes, through the use of in situ hybridization and 3H-thymidine labeling. Effects of PTHrP deficiency on placental calcium transport will be examined. The effects of PTHrP deficiency on the differentiation and activity of parietal endoderm cells that normally synthesize Reichert's membrane will be evaluated in intact embryos and in an established in vitro model system. The PTH gene will also be deleted from the mouse genome to explore the possible overlap in function between PTHrP and PTH; the phenotypes of mice missing each ligand alone will be compared to that of mice missing both ligands. The second Aim is to establish in vivo which actions of PTHrP and of PTH are mediated by the cloned PTH/PTHrP receptor. Mice will be generated missing such receptors through the use of the gene targeting approach. The phenotype of these animals will be compared to that of animals missing one or both ligands. Using these in vivo approaches integrative physiology can be examined in molecular detail. The novel phenotypes of the affected mice may suggest models of analogous human diseases, and the clarification of the normal roles of PTHrP and its receptor may suggest therapeutic strategies for disorders of bone and of mineral metabolism.